The present invention relates to glycosylation and modification of polypeptides, preferably polypeptides of therapeutic value. The administration of glycosylated and non-glycosylated polypeptides for engendering a particular physiological response is well known in the medicinal arts. For example, both purified and recombinant hGH are used for treating conditions and diseases associated with hGH deficiency, e.g., dwarfism in children. Other examples involve interferon, which has known antiviral activity as well as granulocyte colony stimulating factor (G-CSF), which stimulates the production of white blood cells.
The lack of expression systems that can be used to manufacture polypeptides with wild-type glycosylation patterns has limited the use of such polypeptides as therapeutic agents. It is known in the art that improperly or incompletely glycosylated polypeptides can be immunogenic, leading to rapid neutralization of the peptide and/or the development of an allergic response. Other deficiencies of recombinantly produced glycopeptides include suboptimal potency and rapid clearance from the bloodstream.
One approach to solving the problems inherent in the production of glycosylated polypeptide therapeutics has been to modify the polypeptides in vitro after their expression. Post-expression in vitro modification of polypeptides has been used for both the modification of existing glycan structures and the attachment of glycosyl moieties to non-glycosylated amino acid residues. A comprehensive selection of recombinant eukaryotic glycosyltransferases has become available, making in vitro enzymatic synthesis of mammalian glycoconjugates with custom designed glycosylation patterns and glycosyl structures possible. See, for example, U.S. Pat. Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; as well as WO/9831826; US2003180835; and WO 03/031464.
In addition, glycopeptides have been derivatized with one or more non-saccharide modifying groups, such as water soluble polymers. An exemplary polymer that has been conjugated to peptides is poly(ethylene glycol) (“PEG”). PEG-conjugation, which increases the molecular size of the polypeptide, has been used to reduce immunogenicity and to prolong the clearance time of PEG-conjugated polypeptides in circulation. For example, U.S. Pat. No. 4,179,337 to Davis et al. discloses non-immunogenic polypeptides such as enzymes and polypeptide-hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG).
The principal method for the attachment of PEG and its derivatives to polypeptides involves non-specific bonding through an amino acid residue (see e.g., U.S. Pat. No. 4,088,538 U.S. Pat. No. 4,496,689, U.S. Pat. No. 4,414,147, U.S. Pat. No. 4,055,635, and PCT WO 87/00056). Another method of PEG-conjugation involves the non-specific oxidation of glycosyl residues of a glycopeptide (see e.g., WO 94/05332).
In these non-specific methods, PEG is added in a random, non-specific manner to reactive residues on a polypeptide backbone. This approach has significant drawbacks, including a lack of homogeneity of the final product, and the possibility of reduced biological or enzymatic activity of the modified polypeptide. Therefore, a derivatization method for therapeutic polypeptides that results in the formation of a specifically labeled, readily characterizable and essentially homogeneous product is highly desirable.
Specifically modified, homogeneous polypeptide therapeutics can be produced in vitro through the use of enzymes. Unlike non-specific methods for attaching a modifying group, such as a synthetic polymer, to a polypeptide, enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity. Two principal classes of enzymes for use in the synthesis of labeled polypeptides are glycosyltransferases (e.g., sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases), and glycosidases. These enzymes can be used for the specific attachment of sugars which can subsequently be altered to comprise a modifying group. Alternatively, glycosyltransferases and modified glycosidases can be used to directly transfer modified sugars to a polypeptide backbone (see e.g., U.S. Pat. No. 6,399,336, and U.S. Patent Application Publications 20030040037, 20040132640, 20040137557, 20040126838, and 20040142856, each of which are incorporated by reference herein). Methods combining both chemical and enzymatic approaches are also known (see e.g., Yamamoto et al., Carbohydr. Res. 305: 415-422 (1998) and U.S. Patent Application Publication 20040137557, which is incorporated herein by reference).
Carbohydrates are attached to glycopeptides in several ways of which N-linked to asparagine and O-linked to serine and threonine are the most relevant for recombinant glycoprotein therapeuctics. O-linked glycosylation is found on secreted and cell surface associated glycoproteins of all eukaryotic cells. There is great diversity in the structures created by O-linked glycosylation. Such glycans are produced by the catalytic activity of hundreds of enzymes (glycosyltransferases) that are resident in the Golgi complex. Diversity exists at the level of the glycan structure and in positions of attachment of O-glycans to the protein backbones. Despite the high degree of potential diversity, it is clear that O-linked glycosylation is a highly regulated process that shows a high degree of conservation among multicellular organisms.
Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund (1997) Chem. Immunol. 65:111-128; Wright and Morrison (1997) TibTECH 15:26-32). The oligosaccharide side chains of antibodies influence their function (Wittwer & Howard. (1990) Biochem. 29:4175; Boyd et al., (1996) Mol. Immunol. 32:1311) as well as inter- and intra-molecular interactions (Goochee, et al., (1991) Bio/Technology, 9:1347; Parekh, (1991) Curr. Opin. Struct. Biol., 1:750; Hart, (1992) Curr. Opin. Cell Biol., 4:1017; Jefferis & Lund supra; Wyss & Wagner (1996) Curr. Opin. Biotech. 7:409).
For human IgG, the core oligosaccharide usually consists of GlcNAc2Man3 GlcNAc, with slight differences in the numbers of outer residues. For example, variation among individual IgG occurs via attachment of galactose and/or galactose-sialic acid at the two terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc). Removal of the carbohydrate moiety, either by glycosidase cleavage or mutagenesis, has been found to affect binding to C1q and FcγR and the downstream responses such as complement activation and ADCC. (Leatherbarrow et al. Molec. Immunol 22:407-415 (1985); Duncan et al. Nature 332:738-740 (1988); Walker et al. Biochem. J. 259:347-353 (1989)). When the carbohydrate is present, the nature of the sugar residues can influence the IgG effector functions (Wright et al. J. Immunol. 160:3393-3402 (1998)).
Not all polypeptides comprise a glycosylation sequence as part of their amino acid sequence. In addition, existing glycosylation sequences may not be suitable for the attachment of a modifying group. Such modification may, for example, cause an undesirable decrease in biological activity of the modified polypeptide. Thus, there is a need in the art for methods that permit both the precise creation of glycosylation sequences within the amino acid sequence of a polypeptide and the ability to precisely direct the modification to those sites. The current invention addresses these and other needs.